Insulin derivatives with a terminal LPXT motif

ABSTRACT

The present invention relates to insulin derivatives which, in comparison to insulin glulisine and similar derivatives, has a simplified process for synthesis. In particular, the present invention relates to insulin derivatives or physiologically tolerable salts thereof in which the motif Leucine-Proline-X-Threonine appears at the end of the B-chain, where X is any amino acid residue. Due to the nature of the process, the A-chain and B-chain must begin with a Glycine amino acid residue.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable

REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTINGCOMPACT DISC APPENDIX

The file “seqlist.txt”, last modified Dec. 16, 2019 contains thesequence listings referenced in this application and is 1,312 bytes.

SEQ ID No. 1 is a synthetic gene designed to express a single chainpeptide.

SEQ ID No. 2 is the expressed single chain peptide.

SEQ ID No. 3 is the A chain of human insulin.

SEQ ID No. 4 is the B chain of human insulin.

RELATED APPLICATIONS

U.S. Pat. No. 6,221,633B1 April 2001 Ertl et al. US 2015/0118710 A1April 2015 Govindappa et al.

BACKGROUND

The incidence of diabetes mellitus has risen from approximately 108million patients in 1980 to an estimated 422 million in 2014. Meanwhile,cost-related insulin underuse is reported to affect one in four Americanpatients with diabetes. The concurrent advent of insulinrationing-related deaths calls for new, cost-effective approaches to thesynthesis of insulin derivatives.

The present invention relates to insulin derivatives which, incomparison to current derivatives, have a simplified process forsynthesis that relies heavily on recombinant DNA technology. Althoughthere are many novel insulin derivatives that could be created in anumber of hosts by following the process, the present disclosure willfocus on a derivative similar to insulin glulisine expressed in a yeasthost organism, Pichia pastoris. A key aspect of the structure is theLeucine-Proline-X-Threonine motif, where X is any amino acid residue.

The commercial manufacturing process for insulin glulisine requires 15steps that broadly fall under the categories: cell culture and harvest,downstream processing, and final purification. In summary, the insulinglulisine fusion protein is expressed by Escherichia coli and stored ininclusion bodies within the E. coli cells. The fusion protein is foldedand then enzymatically converted during downstream processing. Theproduct of the downstream processing is then purified by chromatography,usually at a separate facility.

The novel use of sortase from Staphylococcus aureus eliminates the needfor downstream processing, can be performed at a single facility, andreduces expenditure for catalysts.

U.S. Pat. No. 6,221,633B1 describes insulin glulisine and alternativefast-acting insulin compounds. The insulin derivatives described byclaim 1 differ from the commercially available insulin glulisine byreplacing phenylalanine (Phe) at position B1 with glycine (Gly), andreplacing threonine (Thr) with leucine (Leu) at position B27 of the Bchain.

US2015/0118710A1 describes a similar process in which the yeast-derivedKexin enzyme creates insulin glargine. However, the limitation of thisprior art is that the B-chain must end with two Arginine amino acidresidues. Recognizing this limitation, the claims of US2015/0118710A1were limited to the long-acting insulin glargine.

SUMMARY

There is provided a formula-specific method for synthesizing functionalinsulin derivatives. The method improves upon the current manufacturingprocess by genetically modifying a yeast, such as Pichia pastoris, or abacteria, such as Escherichia coli, to concurrently express sortase fromthe bacteria Staphylococcus aureus, and a single chain peptidecontaining the sequence Leucine-Proline-X-Threonine, where X is anyamino acid residue.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the modifications to the initial pPIC9K vector forconstitutive expression of sortase. The vector is linearized at the SalIsite prior to electroporation.

FIG. 2 shows the modifications to the initial pGAPZα vector forconstitutive expression of the single chain peptide insulin derivativeprecursor. The vector is linearized at the BglII site prior toelectroporation.

FIG. 3 shows the sites of cleavage for the insulin derivative precursorand the resulting folded structure.

Formula I is used in the claims section to establish relative positionsand bonds of amino acid residues in various insulin derivatives createdby the disclosed process.

DETAILED DESCRIPTION

The object of the present invention is to prepare cost-effective insulinderivatives which after administration, in particular after subcutaneousadministration, have similar properties as commercially availableinsulin derivatives.

It is further an object of the present invention to provide a processfor the preparation of the insulin derivatives having the terminalB-chain motif: Leucine-Proline-X-Threonine, where X represents any aminoacid residue, and in which both the A-chain and B-chain begin withGlycine.

Insulin derivatives are derivatives of naturally occurring insulins,namely human insulin (see SEQ ID No. 3=A-chain of human insulin; see SEQID No. 4=B-chain of human insulin, sequence listing) or animal insulinswhich differ from the corresponding, otherwise identical naturallyoccurring insulin by substitution of at least one naturally occurringamino acid residue and/or addition of at least one amino acid residueand/or organic residue.

Of the twenty naturally occurring amino acids which are universallyencodable, the amino acids glycine (Gly), alanine (Ala), Valine (Val),leucine (Leu), isoleucine (Ile), Serine (Ser), threonine (Thr), cysteine(Cys), methionine (Met), asparagine (ASn), glutamine (Gln),phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and proline (Pro)are designated here as neutral amino acids, the amino acids arginine(Arg), lysine (LyS) and histidine (His) are designated as basic aminoacids and the amino acids aspartic acid (Asp) and glutamic acid (Glu)are designated as acidic amino acids.

Example 3 highlights the necessity of claim 1. This process trivializesthe effort required to create new insulin derivatives as no intermediatecatalysts or processes would need to be developed. As the limitationslaid out in example 3 only differ from human insulin by two amino acidresidues at positions B1 and B27, this method would allow for rapidprototyping of insulin derivatives in parallel cell lines. Once theright formulation is experimentally determined, a different method ofmanufacturing could be devised.

Because Pichia pastoris has recently received recognition for efficientrecombinant expression of proteins, the first example will point to apractical approach of making the two modifications on the same hostorganism. However, it is possible that multiple host organisms couldachieve the same result, if incubated separately.

Example 1

Following the procedure laid out by Zhao et al. published in March of2017, the expression of sortase is achieved extracellularly by modifyingthe commercially available pPIC9K vector to use the GAP Promoter byusing the restriction enzyme sites SacI and BamHI. Then, using thegenetic material of Staphylococcus aureus from cell line ATCC 35556, thegene encoding sortase (SrtA) is inserted to the modified pPIC9K vectorwith the restriction enzymes EcoRI and NotI. The modified vector islinearized by the SalI restriction enzyme to increase transformantexpression of sortase in the yeast, Pichia pastoris. The resultingplasmid is shown in FIG. 1. This modification is selected by growing theyeast on petri dishes with Zeocin agar.

Next, the commercially available pGAPZα vector can be used on theselected transformant Pichia pastoris cell line. SEQ ID No. 1 isprepared synthetically and sent in a plasmid. Once the sequence isisolated from the plasmid with the restriction enzymes EcoRI and NotI,it can be inserted in pGAPZα as shown in FIG. 2. This time, theresulting plasmid is linearized by BglII to increase expressionefficiency and transformant cells are selected with Geneticin.

When the final Pichia pastoris transformant cell line sufficientlyexpresses alpha secretion factor with the single chain peptide from SEQID No. 2, sortase cleaves the single chain peptide as shown in FIG. 3.In FIG. 3, the differences between the resulting insulin derivative andhuman insulin are highlighted as gray amino acid residues.

Example 2

Another embodiment of the disclosed invention would be to expresssortase using Escherichia coli following a procedure similar to the 2017Wu et al. article provided as a supplementary reference. Then, thevector from FIG. 2 could be used to express the precursor peptide inPichia pastoris. The resulting products would need to be extracted andcombined as an intermediate step prior to final filtration. Althoughthis is, in some ways, less efficient than example 1, it could besignificantly more cost effective than the current process for similarinsulin derivatives.

Example 3

The limitation imposed on the formulation of insulin derivatives by theprocess of preceding examples is that each chain of insulin must beginwith Glycine, and the B-chain must end with Leucine-Proline-X-Threonine,where X is any amino acid. While this limitation is unique and specific,there are thousands of potentially viable insulin derivatives. Whenefficient expression of sortase is achieved, as in the first paragraphof example 1, the single chain peptide of SEQ ID No. 2 could arbitrarilybe manipulated at relatively low cost by inserting a marginallydifferent sequence into the commercial pGAPZα vector prior tolinearization.

1. An insulin derivative or a physiologically tolerable salt thereof, inwhich the end of the B-chain is Leucine-Proline-X-Threonine, where Xrepresents any amino acid residue, and in which both the A-chain andB-chain begin with a Glycine amino acid residue.
 2. A process ofexpressing a fully folded functional two chain insulin derivate thatrequires no further processing to make it functionally active, saidprocess comprising steps of i) cloning an insulin derivative peptide andsortase in a yeast, such as Pichia pastoris, or a bacteria, such asEscherichia coli; wherein, the sequence coding for sortase is put underthe control of a constitutive or inducible promoter, ii) co-expressingthe said peptide and sortase; and iii) obtaining a fully functionalinsulin derivative with a Leucine-Proline-X-Threonine motif at theterminal of the B chain, where X is any amino acid residue.
 3. Theprocess as claimed in claim 2, wherein the gene encoding the singlechain insulin derivative peptide is set forth as SEQ ID No. 1, or theexpressed peptide contains SEQ ID No. 2 starting from the fourth aminoacid residue.
 4. The process as claimed in claim 2, wherein the sortasegene was derived from bacteria of the order Lactobacillales or anorganism genetically modified to contain the genetic sequence of aLactobacillales bacteria.
 5. The process as claimed in claim 2, whereinsortase is expressed extracellularly by one host organism, and thesingle chain peptide is expressed separately by another host organism ofthe same species, or of a different species.
 6. A process of convertinga single chain peptide into fully folded biologically active insulinderivative, said method comprising steps of: i) obtaining a host cellcontaining a nucleotide sequence encoding a single chain peptideprecursor of an insulin derivative; ii) combining the single chainpeptide with sortase to convert the insulin derivative precursor into afully folded, biologically active insulin derivative.
 7. An insulinderivative or a physiologically tolerable salt thereof, having theformula I, prepared in accordance with the process of claim 2:

in which (A1-A5) are the amino acid residues in the positions A1 to A5of the A chain of human insulin or animal insulin, (A12-A17) are theamino acid residues in the positions A12 to A17 of the A chain of humaninsulin or animal insulin, A8 and A10 are the amino acid residues inpositions A8 and A10 of the A chain of human insulin or animal insulin,A9 is a serine residue (Ser) or alanine residue (Ala), A19 is Tyr, Pheor Ser, A21 is Asn, Asp, Gly, Ser, Thr or Ala, (B4-B6) are the aminoacid residues in the positions B4 to B6 of the B chain of human insulinor animal insulin, (B8-B18) are the amino acid residues in the positionsB8 to B18 of the B chain of human insulin or animal insulin, (B23-B26)are the amino acid residues in the positions B23 to B26 of the B chainof human insulin or animal insulin, B3 is Arg, Lys, His, Ala or Asn, B20is a glycine residue (Gly) or alanine residue (Ala), B21 is a glutamicacid residue (Glu) or alanine residue (Ala), B22 is an arginine residue(Arg) or alanine residue (Ala), B29 is any naturally occurring aminoacid residue.